Submitted 4 January 2019, Accepted 30 January 2019, Published 26 February 2019
Corresponding Author: Roland Hipol – e-mail – [email protected] 1
The soil fungi producing siderophores of Mt. Yangbew, Tawang, La
Trinidad, Benguet
Hipol RM, Baldelomar JA, Bolinget KC and Solis AFF Department of Biology, College of Science, University of the Philippines Baguio
Hipol RM, Baldelomar JA, Bolinget KC, Solis AFF 2019 – The soil fungi producing siderophores
of Mt. Yangbew, Tawang, La Trinidad, Benguet. Studies in Fungi 4(1), 1–13, Doi 10.5943/sif/4/1/1
Abstract
Siderophores, being iron-chelators, have received much attention in environmental research,
medicine, and biotechnology because of their high affinity and specificity to a wide range of
metals. This study aimed to investigate siderophore production of culturable soil fungi from Mt.
Yangbew, Tawang, La Trinidad, Benguet and to determine their chemical characteristics.
Siderophore production was detected and quantified using Chrome Azurol Sulphonate assay while
the siderophore characterization was through FeCl3, Csáky’s and Arnow’s tests. It was found that
11 out of 20 pure cultures, which showed positive CAS assay results, produce hydroxamates. ITS
primers were used in PCR amplification for the molecular identification of the top-four
siderophore-producers. Based on BLASTn analyses of their sequences, the isolates were found to
be most homologous to species of Talaromyces, Byssochlamys, Paecilomyces, and Penicillium. The
identified species were subjected to phylogenetic analysis using MEGA 7 to the show evolutionary
history and relationship among the fungal species.
Key words – CAS assay – hydroxamates – iron chelators
Introduction
Iron (Fe) as the fourth most abundant element in the terrestrial environment (Drechsel &
Winkelmann 1997) and is greatly needed by most microorganisms in order to perform several
metabolic functions. It is greatly involved in the reduction of oxygen for ATP synthesis, in the
reduction of DNA ribonucleotide precursors, and in the production of heme (Neilands 1995). It is
also a cofactor for several enzymes like ribotide reductase, peroxidase, catalase, nitrogenase,
ribotide reductase, and succinic dehydrogenase (Litwin & Calderwood 1993).
This element exists in two readily inter-convertible oxidation states, Fe (II) and Fe (III). Such
ability to convert between the two states allows iron to play an essential role in numerous electron
transfer processes (Gray & Winkler 1996).
Iron is prevalent in aerobic environments and exists as Fe2 (H2O)63+ but is not readily
available for the uptake of microorganisms because its precipitates as oxyhydroxide polymers
(Guerinot 1994) and, therefore, are insoluble in environmental conditions. Fe (III) is soluble in
water at neutral pH at 10⁻¹⁸ M (Raymond et al. 2003), a concentration far below the concentration
required for growth which is about 10⁻⁷ M in active microbes (Ishimaru & Loper 1993). Iron, as a
result, becomes a growth-limiting factor for many organisms.
In response to this condition, most microorganisms have evolved adaptive mechanisms that
can solubilize iron (Neilands 1995). One such strategy is the production of siderophores, which are
Studies in Fungi 4(1): 1–13 (2019) www.studiesinfungi.org ISSN 2465-4973
Article
Doi 10.5943/sif/4/1/1
2
low molecular weight compounds (600 to 1500 Daltons) that chelate not only iron (Lankford 1973)
but also other essential metals (Johnstone & Nolan 2015) and heavy metals (Braud et al. 2009,
Ruggiero et al. 1999).
Based on the chemical nature of their coordination sites, microbial siderophores may be
classified as hydroxamates, catecholates, carboxylates, or mixed type (Renshaw et al. 2002).
Bacteria secrete siderophores comprising a variety of functional groups (Hider 1984) while most
fungi produce hydroxamate-type siderophores (Leong 1986).
In this study, soli fungi collected from Mt. Yangbew, Tawang, La Trinidad, Benguet was
tested for their capacity to produce siderophores. The types of siderophores secreted were also
investigated. The highest producers of siderophores were identified through molecular means. The
identification of the siderophore-producing fungi will provide baseline data about the presence of
native sources of siderophores for downstream studies relating to several applications that include
agricultural and medical.
Materials & Methods
Collection of Soil Samples
Composite soil sampling was carried out on 18 January 2017 in three locations at the
summit of Mt. Yangbew in Tawang, La Trinidad, Benguet (Fig. 1). The coordinates and elevation
of the sites were determined using a Garmin etrex® 10 GPS.
Location 1–coordinates: 16°27.222' N, 120°36.454' E; elevation: 1652 masl
Location 2–coordinates: 16°27.260' N, 120°36.552' E; elevation: 1657 masl
Location 3–coordinates: 16°27.271' N, 120°36.477' E; elevation: 1647 masl
Fig. 1 – A Map of Luzon Island, Philippines showing the location of Mt. Yangbew relative to the
capital Manila. Map data taken from Google Maps ©2017. B Satellite image of Mt. Yangbew in
perspective view. Image taken from Google Earth ©2017. C Summit of Mt. Yangbew.
The soil samples were obtained using a soil corer and were placed separately in re-sealable
bags. The moisture, temperature, and light intensity of each site were determined using a digital soil
3
analyzer (Model KC300B, Shenzhen Technology Co., Ltd.). The pH of the collected soil sample
was measured using a pH meter.
Media Preparation
In a clean 500 mL Erlenmeyer flask, 15.6 g of Potato Dextrose Agar (PDA) was added to 400
mL of distilled water. To inhibit most bacterial growth, 0.04 g of chloramphenicol was also added.
The mixture was dissolved by thorough stirring and was microwaved oven to fully melt the agar.
The flask was then covered with a cotton plug and the medium was autoclaved for sterilization.
Five mL of the sterile melted medium was poured into sterilized Petri plates. Once the medium has
solidified, the plates were sealed properly and inverted to prevent the accumulation of condensing
moisture on the agar surface. The plates were stored in refrigerator for future use.
For the spread plating, rose Bengal chloramphenicol agar was used. In here, 0.04 g of rose
Bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) was added to 400 mL PDA-
chloramphenicol mixture before sterilization. The presence of the stain suppresses the growth of
bacteria and restricts the size and height of mold colonies (Banks et al. 1985), thereby aiding in the
isolation of slow-growing fungi.
For PDA slants, test tubes covered with cotton plugs, each with 5 mL of prepared medium,
were sterilized. After sterilization, the test tubes were laid across slanting boards, allowing the agar
to solidify. The sterile media slants were refrigerated for future use.
Serial Dilution and Spread Plating
The stock soil suspension was prepared by adding 10 grams of the pooled soil (the mixture of
the three collected soil samples) to 90 mL of distilled water in a clean beaker. The solution was
thoroughly stirred to dispense and evenly distribute the microbial cells. A series of four consecutive
dilutions (Wistreich 2003) was made (10⁻¹, 10⁻², 10⁻³, 10⁻⁴) with 1:9 ratio (1 mL from a lower
dilution was added to 9 mL of distilled water in a separate clean test tube for the next higher
dilution). The four tubes containing the diluted solutions were agitated using a vortex for uniform
distribution of cells.
Dilutions 10⁻², 10⁻³, and 10⁻⁴ were plated into pre-poured PDA plates with Rose Bengal
wherein six media plates were used for each dilution. Using a sterile L-rod, 0.1 mL or 100 μL of
each dilution was spread onto each plate. The plates were properly sealed and were incubated at
30 °C for 48 hours.
Isolation, Purification, and Preparation of Stock Slant Cultures
Morphologically distinct colonies from the inoculated PDA plates were picked and purified
in freshly prepared media plates. Isolated discrete cultures were purified using a single-line streak
plate inoculation for the first purification wherein the plates were incubated at 30 °C for 48 hours.
Grown cultures from these plates were subsequently transferred using an inoculating loop to new
PDA plates without rose Bengal for a second purification, with another 2-day incubation. For stock
cultures of the isolates, fungal growths on plates in second purification were inoculated into PDA
slants using a single-line streak, starting at the bottom of the slanted agar surface and drawing the
inoculating loop towards the topmost part of the agar. The inoculated agar slants were then
incubated at 30 °C for two days.
Siderophore Production Media Preparation
For siderophore production, Grimm–Allen medium (Grimm & Allen 1954) was used. It
contained 1 g K₂SO₄, 3 g ammonium acetate, 3 g K₂HPO₄, 1 g citric acid, and 20 g sucrose in 1 L
of distilled water, and adjusted to pH 6.8 with ammonia (Baakza et al. 2003). The medium was
decontaminated of iron by adding 8-hydroxyquinoline dissolved in chloroform (Messenger &
Ratledge 1985) that, when shaken, formed ferrous or ferric hydroxyquinates. After separation, the
chloroform layer was removed and the medium was washed repeatedly with chloroform to ensure
4
complete removal of iron complexes and any residual 8-hydroxyquinoline, which could inhibit
growth (Messenger & Ratledge 1985).
A 50-mL volume of the medium was dispensed into 500 mL conical flasks. Glassware used
were soaked overnight in 6 mol/L HCl and rinsed with distilled water several times to remove
traces of iron (Schwyn & Neilands 1987). The growth medium was inoculated with an estimated
concentration of 10⁸ fungal spores /mL into flasks and incubated at 28 ± 2 °C. Filtrates of 15-day
old cultures were then subjected to chemical characterization assays.
Chrome Azurol Sulphonate (CAS) Solution Preparation
The production of siderophores by the isolated and purified microorganisms was screened
using the CAS indicator solution (Schwyn & Neilands 1987). The solution was prepared by first
decanting 6 ml of 10 mM hexadecyltrimethylammonium bromide (HDTMA) solution in a 100-mL
volumetric flask. Added to it was a mixture of 1.5 mL iron (III) chloride hexahydrate (FeCl₃·6H₂O)
and a 7.5 ml of 2 mM aqueous CAS dye solution were added in the flask. A buffer solution was
then prepared by dissolving 4.307-g 1,4-piperazinediethanesulfonic acid (Pipes) in 30 mL distilled
water, adjusting its pH to 5.6 using sodium hydroxide (NaOH) before adding it to the solution.
Lastly, to get a precise measurement of the solution, distilled water was poured slowly until the
base of meniscus matched with the marked line in the volumetric flask (Yeole et al. 2001).
Detection and Quantification of Siderophore Production
Detection and quantification of siderophore production was based on Fekete et al. (1989)
methodology, where 150 µL of supernatant (15 day-old fungal cultures in Grim-Allen media) was
added to 150 µL CAS assay solution, obtaining a 1:1 ratio. A color change from blue to orange
occurred which indicated the removal of iron from the dye (ternary) complex indicator (Barton &
Hemming 1993).
A filter-based multimode microplate reader (FLUOstar® Omega, BMG LABTECH) was
used to detect the absorbance of the samples at 630 nm. The CAS reagent was used as the blank
solution. The amount of siderophore produced was computed in terms of their percentage
siderophore units (% SU) using the formula of Tailor & Joshi (2012; Equation 1), where Ar is the
absorbance of the CAS reagent and As is the absorbance of each sample, all at 630 nm. This was
done in triplicate and the standard error of the means was calculated.
(Equation 1)
The assay revealed the top four siderophore-producing fungi from the soil sample and was
subjected to morphological and molecular identification.
Detection of Hydroxamate Siderophores
Presence of hydroxamate-type siderophores was determined using the quantitative Iron
Perchlorate Test and the confirmatory colorimetric Csáky Assay.
Iron Perchlorate Test To 1 mL of culture supernatant, 1-5 mL of freshly prepared aqueous 2% FeCl₃ solution was
be added. The coordination of the metal ion with the siderophore yielded an orange color. The
absorbance was measured between 200-600 nm and a peak at 420-450 nm indicated the presence of
an orange-colored ferric hydroxamate (Atkin et al. 1970, Neilands 1981).
Csáky Assay
A 1-mL amount of culture supernatant was placed in test tube and was hydrolyzed with 1 mL
of 6 N H₂SO₄ in a boiling water bath for 6 hours. The solution was then buffered by adding 3 mL
35% NaOAc solution and was followed by the addition of 0.5 mL I2 solution (1.3 g iodine per 100
5
mL glacial acetic acid). After 3-5 minutes, excess I2was destroyed with 1 mL Na3AsO4 solution.
One mL α-naphthylamine solution was added thereafter and color was allowed to develop. A wine
red color indicated the presence of a hydroxamate type of siderophore (Adhikari et al. 2013, Ali &
Vidhale 2013, Csáky 1948).
Detection of Catechol Siderophores
The occurrence of catechol-type siderophores were detected using the quantitative Iron
Perchlorate Test and the confirmatory colorimetric Arnow’s Test.
Iron Perchlorate Test
Catecholate siderophores form a wine-colored complex with amM≈ 5 at 495 nm on addition of
ferric chloride. To 1 mL of culture supernatant culture, 1 mL of 2% FeCl₃ was added. The
formation of a wine-colored complex was monitored at 495 nm (Neilands 1981).
Arnow’s Test
One mL culture supernatant was placed in a screw-capped tube and added with 1 mL nitrite-
molybdate reagent (containing 10 g each of NaNO2 and Na₂MoO₄‧2 H₂O in 50 mL water) and 1 ml
0.5 N HCl (a yellow-colored solution at this point). Finally, 1 mL 1 N NaOH solution was added
and red color indicates the presence of the catecholate type of siderophore (Jikare & Chavan 2013,
Arnow 1937).
Morphological Analysis
Pure cultures of the top four siderophore-producing fungi were inoculated on the center of the
Petri plates (9 cm diameter) containing PDA media with chloramphenicol (200 µg/ml) and
incubated for 3 days. The macroscopic features of the four species were described including the
height and width (in cm), the color, and the growth characteristics such as surface, margin,
elevation and form of the whole. These morphological characteristics were observed under a stereo
zoom microscope (Model: SZM-B, OPTIKA Accessories).
Molecular Identification and Phylogenetic Analysis
The top four siderophore-producing isolates were sent to the sequencing laboratory of
Macrogen, Inc. in Geumcheon-gu, Seoul, South Korea for precise genomic DNA extraction, PCR
amplification, PCR purification, bidirectional sequencing, and BI report. The specific DNA
sequence amplified was the ITS (internal transcribed spacer) region, with ITS1
(TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) as the forward and
reverse primers, respectively. Macrogen’s sequencing analysis made use of a 3730xl DNA
Analyzer (Applied Biosystems™).
Information on the resultant primary biological sequences were then compared to deposited
nucleotide DNA sequences in public databases using the National Center for Biotechnology
Information’s Basic Local Alignment Search Tool (BLAST), specifically the nucleotide–nucleotide
BLAST (blastn), to determine the most similar DNA sequences from the database to the fungal
species positive for siderophore production (Altschul et al. 1990). The ITS sequences were placed
within tentative taxa by determining the sequence from the GenBank database it is most
homologous to.
Multiple sequence alignments of ITS sequences were performed using ClustalW.
Phylogenetic analysis was conducted through MEGA7: Molecular Evolutionary Genetics Analysis
version 7.0 for bigger datasets by Maximum Likelihood method and the evolutionary history was
inferred using Kimura 2-parameter model (Kimura 1980). A discrete Gamma distribution was used
to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4699)).
Statistical significance of the tree branches was evaluated by bootstrap analysis based on 1000
bootstrap replicates.
6
Results
Twenty isolates of distinct fungal colonies were cultured from the soil sample of Mt.
Yangbew in Tawang, La Trinidad, Benguet. Purified cultures were designated as isolate BBS-001
to isolate BBS-020. Siderophore production was determined and those that showed a positive result
were subjected to a comprehensive screening.
Detection of Siderophore Production
Out of 20 isolates, only 10 showed a positive result in the CAS assay, the universal test for
the detection of siderophores (see Fig. 2). These isolates, BBS-001, BBS-003, BBS-004, BBS-005,
BBS-006, BBS-007, BBS-008, BBS-0015, and BBS-0019, were subjected to several assays that
determined the type of siderophore they produced.
Furthermore, Fig. 2 show that six isolates exhibited more than 90% SU, the highest being
BBS-008 with 97.91% SU, while the four others were below 70% SU, with the lowest at 47.38%
SU being BBS-004. These four isolates with the highest siderophore units produced were subjected
for molecular identification. Following the top producer BBS-008 was BBS-006 with 97.73% SU,
BBS-019 ranked third with 92.76% SU, and BBS-005 ranked fourth with 92.32% SU produced.
Chemical Characterization of Siderophores
Qualitative tests were performed in conjunction with the quantitative UV spectrophotometric
methods to evaluate the production of siderophores by the isolated rhizosphere fungi. Nine out of
the 10 siderophores tested positive in the detection for hydroxamate-type (Table 1). They recorded
a maximum absorbance between 420 nm – 450 nm in the FeCl3 test and between 420 nm – 495 nm
in the Csáky assay. Other authors have considered a wine-red color development in the latter as
indicative for hydroxamate production, even with no spectrophotometric analysis.
Only BBS-004 and BBS-005 gave positive results in both the tests that detected N-
hydroxylated amine bonds with peaks at 432 nm and 440 nm, respectively, in the FeCl3 test. Isolate
BBS-001 exhibited maximum absorbance at 424 nm, BBS-007 at 438 nm, and BBS-009 had
multiple peaks (424 nm, 428 nm, 438 nm, and 448 nm) within the desired range of absorbance.
These three, however, failed to show a positive result in the Csáky assay. Isolate BBS-003
developed a wine-red color in the Csáky assay and this was supported by having multiple
maximum absorbance at 420 nm, 423 nm, 432 nm, and 440 nm. A similar occurrence was also
observed for BBS-015 where four absorbance peaks (422 nm, 432 nm, 446 nm, and 450 nm) were
noted. Isolates BBS-006, BBS-008, and BBS-019 all developed a wine-red color. BBS-002 failed
to show positive results in tests for both hydroxamate and catecholate tests.
Table 1 Detection of the chemical nature of siderophores
Isolate
Hydroxamate Catecholate
FeCl3 Test
(420 nm-450 nm)
Csáky Assay
(420 nm-495 nm)
FeCl3 Test
(420 nm-450 nm)
Arnow’s Test
(515 nm)
BBS-001 - - - -
BBS-003 - + - -
BBS-004 + + - -
BBS-005 + + - -
BBS-006 - + - -
BBS-007 + - - -
BBS-008 - + - -
BBS-009 + - - -
BBS-015 - + - -
BBS-019 - + - -
** (+) – positive
(-) – negative
7
Fig. 2 – Quantification of siderophore production by fungal isolates (in % siderophore units, SU).
Asterisks after the data labels denote that the isolate belong to the top four producers.
Morphology and Identification of Siderophore-Producing Fungal Isolates
The fungal isolates were subjected to colony morphology for preliminary identification.
There were noticeable differences in the morphological characteristics of the colonies of the
isolates (Table 2), specifically on their appearance in PDA medium, size (in mm), color, reverse
color, surface of mycelial texture, margin, elevation, and form. Conidial area of BBS-008 were
yellow orange, while the other three all had white margins with either brown or bluish centers. All
isolates exhibited a circular colony form except BBS-005 which was irregularly shaped. The
colonies of BBS-008 and BBS-005 were both wrinkled while those of BBS-006 and BBS-019 were
dry and powdery.
Along with colony morphology, the isolates were subjected to molecular characterization.
DNA sequences showing ≥98% similarity was considered to belong to the same species (Arnold &
Lutzoni 2007) while sequences showing ≥95% similarity were considered to belong to the same
genus. Sequences that showed <95% similarity were considered unidentified taxon. Table 3 shows
the nearest matches for the two ITS sequences to deposited strains in the database.
Isolate BBS-008, which has the maximum siderophore production, was most homologous
with a strain of Talaromyces tardifaciens, with maximum identity at 96%. This was also the
percentage for the nearest match of BBS-006, which is Byssochlamys nivea. Putative taxonomic
affinities for isolates BBS-019 and BBS-005 were Paecilomyces formosus (96%) and Penicillium
citrinum (99%), respectively.
The phylogenetic affinities of the fungal isolates (Fig. 3) were determined through
phylogenetic analysis using MEGA7. Evolutionary history was inferred using the Maximum
Likelihood method based on the Kimura 2-parameter model (Kimura 1980) with a discrete Gamma
distribution to model evolutionary rate differences among sites [5 categories (+G, parameter =
0.4699)]. The consensus tree was constructed from fifteen aligned nucleotide sequences–11
reference sequences obtained through BLASTn search and the four isolates’ sequences—with 1000
bootstrap replications. The reference sequences selected were those that had the highest sequence
percentage homology to the queries. The siderophore-producing zygomycete fungi
Cunninghamella elegans served as the outgroup.
Discussion
Detection and characterization of siderophores
The screening of 20 fungal isolates cultured from the soil of Mt. Yangbew, Tawang, La
Trinidad, Benguet by the CAS assay and FeCl3 test demonstrated the widespread occurrence of
siderophores in fungi in iron uptake. The high levels of siderophores produced, especially of the top
four isolates, were indicative that the fungal species were highly adapted to iron-unavailable
conditions.
8
Among the isolates successfully grown in the GA medium, six out of 10 recorded >90% siderophore production while four were below 70%. It
is only logical that there are differences in the quantity of siderophores produced by different microorganisms. Reigh & O’Cannell (1988), Roy et al.
(1994) claimed reports of strain-to-strain variation in siderophore production.
All the siderophores secreted by the fungal isolates were detected as hydroxamates and no catecholate-type was detected, validating reports of
their usual occurrence only in bacteria (Dave & Dube 2000). Catecholate siderophores have been reported in fungi but their occurrence is rarer. A
marine fungus, Penicillium bilaii, was found to produce pistillarin, a rare catechol siderophore, when grown under relatively high-iron conditions
(Capon et al. 2007).
Table 2 Colony morphology of top four siderophore-producing fungi isolated from the rhizosphere of Mt. Yangbew
Fungal Isolate Colony
Appearance
Size (mm) Color Reverse Color Surface Margin Elevation Form
BBS-008
18 × 16 Yellow orange Orange Wrinkled Entire Umbonate Circular
BBS-006
44 × 44 Brown center,
white margin
Yellow with
white margin
Dry, powdery Entire Raised,
spreading edge
Circular
BBS-019
40 × 40 Brown center,
white margin
Yellow with
white margin
Dry, powdery Entire Raised,
spreading edge
Circular
BBS-005
17 × 15 Blue green with
white margin
White Wrinkled Lobate Wrinkled
convex
Irregular
9
Table 3 Putative taxonomic affinities of sequenced types inferred from BLASTn searches of ITS
sequences
Isolate ITS1 Nearest
Match
Max Identity
(%)
ITS4 Nearest
Match
Max Identity
(%)
Identity
BBS-008 Talaromyces
loliensis CBS
643.80
(KF984888.1)
94% Talaromyces
tardifaciens CBS
250.94
(KF984874.1)
96% Talaromyces
sp
BBS-006 Byssochlamys
fulva CBS 146.48
(NR_103603.1)
92% Byssochlamys
nivea MUCL
39714
(DQ464362.1)
96% Byssochlamys
sp
BBS-019 Paecilomyces
formosus CBS
990.73B
(FJ389929.1)
96% Paecilomyces
formosus CBS
990.73B
(FJ389929.1)
96% Paecilomyces
sp
BBS-005
Penicillium
citrinum NRRL
1841
(NR_121224.1)
96% Penicillium
citrinum NRRL
1841
(NR_121224.1)
99% Penicillium
citrinum
The production of hydroxamate siderophores has been reported in numerous fungi starting
with the first report of siderophore from Ustilago sphaerogena (Winkelmann 1992). In terms of
possible applications, hydroxamate siderophores are better than catecholates for agriculture because
they are comparatively stable, have high iron-chelating abilities (Kloepper et al. 1980), and impart
suppressiveness to the soil, which is important for preventing the growth of phytopathogens
(Mazzola 2002).
Molecular Identification of Siderophore-Producing Fungi
The internal transcribed spacer (ITS) region, which contains the 5.8S gene and is located
between the 18S and 26S rRNA genes, has the highest probability of successful identification for
the broadest range of fungi, with the most clearly defined barcode gap between inter- and
intraspecific variation (Schoch et al. 2012). Deducing the relation of the isolate sequences to
reference sequences (Table 3) was based on Arnold & Lutzoni (2007) suggested threshold cut off
for genus and species acceptance where a 95% sequence similarity for the ITS region is considered
as a conservative species boundary.
The occurrence of the four fungal genera in the soil is expected as they have been isolated
from soils beforeTalaromyces is a genus of about 25 species, also mostly soil inhabiting and is
typical in warmer climates (Pitt & Hocking 1997). The Penicillium genus is diverse in terms of
numbers of species and range of habitats.
Penicillium citrinum has been reported to secrete iron-chelators (Baakza et al. (2003). Five
new coprogen-type hydroxamate siderophores were discovered from an Australian mud dauber
wasp-associated unspecified species of Talaromyces (CMB-W045), along with three others that
were first detected in other fungal isolates (Kalansuriya et al. 2017). Paecilomyces variotii was
found to produce both hydroxamate and carboxylate siderophores, suggesting that carboxylates
could also be present outside Mucorales (Vala et al. 2000). Other species of Paecilomyces were
also reported to produce hydroxamate siderophores—P. lilacinus and Paecilomyces sp. AB7
(Baakza et al. 2003). For Penicillium, P. resticulosum (Konetschny-Rapp et al. 1988), P.
funiculosum, P. oxalicum, and P. fellutanum (Baakza et al. 2003) all tested positive for
hydroxamate-type siderophores. It is only the genus Byssochlamys that has not been reported to
produce siderophores.
10
Fig. 3 – Phylogenetic tree by Maximum Likelihood method showing the relationship among fungal
ITS sequences from the soil microbiota of Mt. Yangbew in La Trinidad, Benguet with reference
sequences obtained through BLAST. The evolutionary history was inferred by using the Maximum
Likelihood method based on the Kimura 2-parameter model (Kimura 1980). Initial tree(s) for the
heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a
matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL)
approach, and then selecting the topology with superior log likelihood value. A discrete Gamma
distribution was used to model evolutionary rate differences among sites (5 categories (+G,
parameter = 0.3816)). Bootstrap values (1000 replicates) are shown at the nodes. The tree is drawn
to scale, with branch lengths measured in the number of substitutions per site. There was a total of
321 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al.
2016).
Phylogenetic analyses showed that the sequenced soil isolates belonged to two subclades
under the Phylum Ascomycota, which is the largest within the kingdom Fungi. Isolates BBS-006
and BBS-019 fall under Sordariomycetes while BBS-008 and BBS-005 belong to Eurotiomycetes.
BBS-005 was found to be closely related to Penicillium citrinum with 99% similarity (Table
3) and supported by a high bootstrap value of 99 (Fig. 3). Its affinity to the siderophore producer P.
chrysogenum (not reference sequence) was also well-supported at a 94-bootstrap value, suggesting
that the isolate is indeed Penicillium citrinum. The phylogenetic placement of BBS-008 clustering
with T. tardifaciens (96% similarity to ITS4 according to BLASTn) was also highly-supported
(bootstrap value is 94). Its affiliation, however, to the nearest match for its ITS1 sequence (T.
loliensis) was not supported at 23% bootstrap value.
The consensus tree shows that the three reference Byssochlamys species and BBS-019—
whose identity as P. formosus was well-supported by the 86% bootstrap value—was found to
branch out from BBS-006, which was most homologous to B. nivea (96% similarity to its ITS
sequence). The nearest phylogenetic neighbor of the BBS-006 was Paecilomyces, indicating that it
may be more related to it than its reference species. Such divergence may be explained by Stolk &
Samson (1971) who revised the genus Byssochlamys with the discovery of it having Paecilomyces
anamorphs, which, in contrast to the former’s sexual state, are asexual.
Conclusion
This study demonstrated strain-to-strain differences in the production of siderophores. The
seven fungal isolates having more than 90% SU imply that they are well-adapted to an iron-limited
Eurotiom
ycetes S
ordariomycetes
11
conditions than those below 90%, confirming that siderophore-mediated iron uptake is important to
rhizosphere fungi in Mt. Yangbew, Tawang, La Trinidad, Benguet. Also, fungi from the collection
site predominantly produce hydroxamate siderophores.
Siderophores of the identified fungal isolates have been reported, except for Byssochlamys.
This study is the first to present evidence of production of hydroxamate siderophores in this genus.
For future biotechnological applications, the top four identified siderophore-producing fungi
from Mt. Yangbew, Tawang, La Trinidad, Benguet may be considered. Possible applications
include bioremediation of heavy metal pollution, biological control of microbial plant pathogens, as
well as biomedical studies. The elucidation of the chemical structure of the siderophores is also a
promising endeavor, especially which of BBS-06 identified as Byssochlamys sp.
Acknowledgements
The authors would like to acknowledge the support of the Department of Biology, College of
Science, University of the Philippines Baguio for the conduct of this study.
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